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AntimiR caused increased expression of miRNA


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#1 netdavid

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Posted 21 May 2010 - 05:44 AM

Hello everybody,
I have a serious problem that I hope somebody can help me to solve. I performed an experiment to knock down a specific miRNA. My experimental design included a control (without any antimir, just my cells), an antimir negative control (Cy-3 labeled) and an antimir against the miRNA of interest. After 24 hours from transfection (I used lipofectamine), I checked the transfection efficiency on my antimir negative control Cy-3 labeled (by fluorescence microscopy) which was near to 90%. From the different samples I extracted total RNA using mirVana kit and I retrotranscribed and quantified by real-time PCR my miRNA of interest to check the effective knock down....... now the problem is that I have about 20 times more miRNA of interest in the antimir treated as compare to the antimir control. Furthermore both the antimir negative control and the antimir of interest treated samples have an higher content of my miRNA of interest as compared with the sample without transfection. So what's happening? Do I made an error somewhere? Any advice will be very appreciated. Thank you in advance.

David

#2 Fizban

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Posted 25 May 2010 - 11:09 PM

Hello everybody,
I have a serious problem that I hope somebody can help me to solve. I performed an experiment to knock down a specific miRNA. My experimental design included a control (without any antimir, just my cells), an antimir negative control (Cy-3 labeled) and an antimir against the miRNA of interest. After 24 hours from transfection (I used lipofectamine), I checked the transfection efficiency on my antimir negative control Cy-3 labeled (by fluorescence microscopy) which was near to 90%. From the different samples I extracted total RNA using mirVana kit and I retrotranscribed and quantified by real-time PCR my miRNA of interest to check the effective knock down....... now the problem is that I have about 20 times more miRNA of interest in the antimir treated as compare to the antimir control. Furthermore both the antimir negative control and the antimir of interest treated samples have an higher content of my miRNA of interest as compared with the sample without transfection. So what's happening? Do I made an error somewhere? Any advice will be very appreciated. Thank you in advance.

David

That's kind of interesting.
In my opinion you should start with simple things first.
First simple assumption usually is that u made a mistake.
U sure you ordered complementary sequence and not the mature seq? (i know it sounds very stupid but CAN happen!!!)
try again the same exp (if u were speaking about n=1). Id use at least 3 different concentrations of anti miR and see if u see a dose-response in terms of overexpression. was your control just cells or cells with lipofectamine? why did you check transfection efficiency only in negative control?
second: which inhibition system did u use? difficoult to help you without knowing. also try supplier's tech support.
third: u need to have a functional test (reporter assay on at least a target of your miR) to check what happens
4th: maybe u did everything well and it's not an artifact: could be interesting, maybe a regulatory loop answering to your inhibition. what if u block transcription?

let us know!!
bye
Fiz

Edited by Fizban, 25 May 2010 - 11:11 PM.


#3 netdavid

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Posted 25 May 2010 - 11:51 PM

Thanks Fizban,
to answer your question:
First: I checked the sequence and it should be ok. the control was treated with lipofectamine. So far I tried only the highest antimir concentration proposed by Ambion, but I tried different time point 24 and 48 hours after transfection. The effect is stronger at 24 hours and much weaker at 48. I check the efficiency only in the control because only in this one I have the Cy3 dye conjugated with the mock antimir.
Second: I contacted the Tech support I didn't received an answer yet.
Third: I just ordered an antibody to check the presence of one of the miRNA target.
4th. That's a good idea. How could I block the transcription?? A regulatory loop is a possibility...

Finally..... I found an interesting paper published last year: "Potent inhibition of microRNA in vivo without degradation", Scott Davis et al, Nucleic Acid Research 2009. They showed that there are multiple mechanisms of miRNA inhibition by ASOs. I'm wondering if really my miRNA is degraded or just abducted by the antimir. This could explain everything because although it is bloked it could be amplified. I'm also wondering whether the realtime PCR primers can amplify the antimir.....

Thanks again

#4 Samantha

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Posted 26 May 2010 - 07:04 AM

Dear David,

I had the same problem as yours. My case was I tried to knock down a specific miRNA by transfecting LNA miRNA inhibitor and measured the miRNA expression by TaqMan real-time PCR. It turned out my miRNA expression was even higher. I gave the data to the company and they replied there were two possibilities. One was there was a feedback loop, i.e. knock down of the miRNA would force the cells to produce more that specific miRNA. Another possibiliy was it's the incompatibility of LNA miRNA inhibitor and the RT system of ABI. It's a bit complicated, but what I remember the salesman told me was because of the design of the probes and stem loop based RT reaction, the miRNA inhibitor would act as a template for the RT reaction eventually (sorry that I forgot the details) and thus the expression of the miRNA would become higher. To overcome this problem, the company suggested me to use both the RT system and the miRNA inhibitor offered by their company or use other methods like reporter assay to assess the transfection efficiency instead of simply by real-time PCR. Hope this information can help you.

Samantha

Hello everybody,
I have a serious problem that I hope somebody can help me to solve. I performed an experiment to knock down a specific miRNA. My experimental design included a control (without any antimir, just my cells), an antimir negative control (Cy-3 labeled) and an antimir against the miRNA of interest. After 24 hours from transfection (I used lipofectamine), I checked the transfection efficiency on my antimir negative control Cy-3 labeled (by fluorescence microscopy) which was near to 90%. From the different samples I extracted total RNA using mirVana kit and I retrotranscribed and quantified by real-time PCR my miRNA of interest to check the effective knock down....... now the problem is that I have about 20 times more miRNA of interest in the antimir treated as compare to the antimir control. Furthermore both the antimir negative control and the antimir of interest treated samples have an higher content of my miRNA of interest as compared with the sample without transfection. So what's happening? Do I made an error somewhere? Any advice will be very appreciated. Thank you in advance.

David



#5 Fizban

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Posted 26 May 2010 - 11:03 PM

Thanks Fizban,
to answer your question:
First: I checked the sequence and it should be ok. the control was treated with lipofectamine. So far I tried only the highest antimir concentration proposed by Ambion, but I tried different time point 24 and 48 hours after transfection. The effect is stronger at 24 hours and much weaker at 48. I check the efficiency only in the control because only in this one I have the Cy3 dye conjugated with the mock antimir.
Second: I contacted the Tech support I didn't received an answer yet.
Third: I just ordered an antibody to check the presence of one of the miRNA target.
4th. That's a good idea. How could I block the transcription?? A regulatory loop is a possibility...

Finally..... I found an interesting paper published last year: "Potent inhibition of microRNA in vivo without degradation", Scott Davis et al, Nucleic Acid Research 2009. They showed that there are multiple mechanisms of miRNA inhibition by ASOs. I'm wondering if really my miRNA is degraded or just abducted by the antimir. This could explain everything because although it is bloked it could be amplified. I'm also wondering whether the realtime PCR primers can amplify the antimir.....

Thanks again

That's interesting. Maybe you could try cotransfection of a plasmid with GFP or another tracer to assess efficiency. I'd try a direct approach to test inhibition, maybe the reporter appreach in addition to WB. If your miRNA of interest is well known maybe you'll find someone who can share an already tested construct. I have a couple of constructs (contact me by PM if interested). try also using the forum to ask or go to ADDGENE website, a noprofit organization who receive plasmids from labs who are willing to share at very low prices!
you could try Actinomycin D to block transcription but maybe there are better ways.
Samantha has a good and interesting point too....distributors sometimes forget to tell things..... :)
maybe you should try another way of inhibition
let us know
Fiz

#6 netdavid

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Posted 26 May 2010 - 11:47 PM

Thank you guys,

Samantha, the fact that you had the same problem make me less frustrated..... :) . I really think that there is a problem in the ABI system. I did a time course study using the antimir and the effects on the miRNA of interest decrease in time. This could be due to a degradation of the complex antimir/miRNA.... so less template/antimir to amplyfy at 72 hours.

Fizban, actually I was thinking to use a reporter approach to test the inhibition, but so far I didn't find any tested construct.

I will let you know.

#7 netdavid

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Posted 02 June 2010 - 01:19 AM

Dear friends and collegues, finally I received an answer from the technical support, I will post it because I think could help others users:
________________________

Dear David,

Yours is a very good question and one that has been raised a number of times. However, the answer is not as simple or direct.

The anti-miRs work by targeting complementary endogenous mature miRNA sequences and through their chemical modification prevent those miRNAs from being processed by the RISC in the cell. This effectively blocks their intended action toward their expected target. Although anti-miRs do not reduce the level of absolute expression of miRNAs in the cell, the apparent amount of mature miRNAs that is measured by Taqman microRNA assays will be reduced. (but we donít always see a reduction in endogenous miRNA level when anti-miRs are used). Indeed the cycling process of the PCR will make the anti-mir dissociate from the target miRNA and expression would still be visible.

The recommended way to measure the action of anti-miRs is to measure its effect through the expression of the miRNA target(s), preferrably at the protein level as miRNAs mostly act at the translational level without affecting the transcripts. Taqman miRNA assays are better suited for quantifying original endogenous miRNAs expression in the absence of exogenous RNA molecules.

Having said that, Taqman miRNA assays can also be used to quantify transfected pre-miRs (miRNA mimics) in the cells. Pre-miRs are taken up by RISC the same way endogenous miRNAs are and resulting mature miRNA is presented to its corresponding targets. The assays quantify the amount of mature miRNA resulting from the pre-miR or miRNA processing.

I hope you find this information of help

If you have any further questions, please do not hesitate to contact me again

Kind Regards

_____________________




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